DNA branch migration, a process whereby two homologous DNA duplexes exchange strands, is an essential component of genetic recombination. The elementary step of branch migration is movement of the Holliday junction one base pair in either direction with equal probabilities; thus branch migration can be modeled as a random walk. We previously demonstrated that a single base mismatch is sufficient to block nonenzymatic branch migration. Since homologous recombination regularly involves the exchange of DNA strands between two similar but not identical duplexes, our result suggested that recombination proteins are required to facilitate branch migration through sequence heterology. In order to further characterize the branch migration step, we developed an assay to determine kinetic parameters of spontaneous branch migration. We have measured the step time of branch migration, that is the time required for the Holliday junction to move one base pair, as a function of temperature and ionic conditions. The rate of branch migration is even faster. We attribute the effect of metal ions on the rate of branch migration to the effect these metals have on the conformation of the four-armed Holliday junction. Subtle differences in the conformation of the junction can have a tremendous effect on the overall rate of branch migration. Our results establish that spontaneous branch migration in the presence of Mg2+ is too slow to account for heteroduplex formation in biological systems and underscore the importance of proteins in promoting branch migration during recombination. We are also interested in characterizing eukaryotic enzymes involved in recombination and DNA repair. We have obtained a partial cDNA clone from chicken with extensive sequence homology to yeast and human type I DNA ligases.